RESEARCH ARTICLE
White Pitaya (Hylocereus undatus) Juice Attenuates Insulin Resistance and Hepatic Steatosis in Diet-Induced Obese Mice Haizhao Song1,2, Zihuan Zheng1, Jianan Wu1, Jia Lai1, Qiang Chu1, Xiaodong Zheng1,2* 1 College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, P. R. China, 2 Fuli Institute of Food Science, Zhejiang University, Hangzhou, P. R. China * [email protected]
Abstract
OPEN ACCESS Citation: Song H, Zheng Z, Wu J, Lai J, Chu Q, Zheng X (2016) White Pitaya (Hylocereus undatus) Juice Attenuates Insulin Resistance and Hepatic Steatosis in Diet-Induced Obese Mice. PLoS ONE 11 (2): e0149670. doi:10.1371/journal.pone.0149670 Editor: Jonathan Peterson, East Tennessee State University, UNITED STATES Received: September 18, 2015 Accepted: February 1, 2016 Published: February 25, 2016 Copyright: © 2016 Song et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Key Technology R&D Program of China (Grant No.2012BAD33B08, http://program.most.gov.cn/, received by XDZ), Zhejiang Provincial Natural Science Foundation of China (Grant No. Z14C200006, http://www.zjnsf.gov.cn/, received by XDZ) and the Foundation of Fuli Institute of Food Science, Zhejiang University (Grant No. KY201301, http://fifs.zju.edu.cn/content/detail.php?sid=3&cid= 642, received by XDZ). The funders had no role in
Insulin resistance and hepatic steatosis are the most common complications of obesity. Pitaya is an important source of phytochemicals such as polyphenols, flavonoid and vitamin C which are related to its antioxidant activity. The present study was conducted to evaluate the influence of white pitaya juice (WPJ) on obesity-related metabolic disorders (e.g. insulin resistance and hepatic steatosis) in high-fat diet-fed mice. Forty-eight male C57BL/6J mice were assigned into four groups and fed low-fat diet with free access to water or WPJ, or fed high-fat diet with free access to water or WPJ for 14 weeks. Our results showed that administration of WPJ improved high-fat diet-induced insulin resistance, hepatic steatosis and adipose hypertrophy, but it exerted no influence on body weight gain in mice. Hepatic gene expression analysis indicated that WPJ supplement not only changed the expression profile of genes involved in lipid and cholesterol metabolism (Srebp1, HMGCoR, Cpt1b, HL, Insig1 and Insig2) but also significantly increased the expression levels of FGF21-related genes (Klb, FGFR2, Egr1 and cFos). In conclusion, WPJ protected from diet-induced hepatic steatosis and insulin resistance, which was associated with the improved FGF21 resistance and lipid metabolism.
Introduction Type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) are common consequences of a combination of metabolic disorders such as dyslipidemia, hepatic steatosis, glucose intolerance and insulin resistance [1]. Mounting evidence suggests that insulin resistance and hepatic steatosis, the most common complications of obesity, are characterized by impaired energy metabolism and attributed by complex interactions between genetic background and environmental impacts such as dietary and sedentary habits[2–6]. Moreover, insulin resistance is often accompanied by hepatic steatosis [7]. Traditional pharmacotherapies for type 2 diabetes and NAFLD are often accompanied by side effects. Recently, growing evidence has suggested that natural bioactive compounds in food exerts many beneficial effects on obesity and its metabolic consequences [8–10].
PLOS ONE | DOI:10.1371/journal.pone.0149670 February 25, 2016
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White Pitaya Juice Attenuates Insulin Resistance and Hepatic Steatosis
study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Pitaya, also known as dragon fruit, is among the most important commercial tropical fruits around the world, and three varieties have been classified as Hylocereu polyrhizus (red pitaya), Hylocereus undatus (white pitaya) and Hylocereus megalanthus (Yellow pitaya) [11]. Pitaya is an important source of phytochemicals such as polyphenols, flavonoid and vitamin C which are related to its antioxidant activity [12, 13]. Especially the red and white pitaya have recently drawn growing attention worldwide not only because of their economic values but also their health benefits [14]. Red pitaya consumption was reported to decrease total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels while increasing the high-density lipoprotein cholesterol (HDL-C) levels in type 2 diabetic subjects. The similar lipid-improving effect of its menthol extract was found in high-fat diet-fed rats [15, 16]. In addition, the antidiabetic effect of red pitaya has also recently been demonstrated. For instance, red pitaya significantly improved insulin resistance in rats and 600 g amount of red pitaya fruit consumption every day decreased the blood glucose level in type II diabetic subjects [15, 17]. Ramli et al. also reported that supplementation of red pitaya juice could induce a trend of glucose and uric acid normalization associated with reduced ALP and ALT but increased AST levels in high-carbohydrate and high-fat diet-fed rats [18].Though both red and white pitaya are reported to be rich, natural and cost-effective source of bioactive nutrients, few studies focused on the beneficial effects of white pitaya on diabetes and NAFLD. The objective of the present study was to investigate the influence of white pitaya juice (WPJ) on obesity-related hepatic steatosis and insulin resistance in high-fat diet-induced obese mice and explore the underlying mechanism by which pitaya juice exerted its beneficial effects. To the best of our knowledge, this is the first study to evaluate the effect of white pitaya on type II diabetes and NAFLD.
Materials and Methods Chemical reagents and juice preparation The solvents and reagents were obtained from Aladdin (Shanghai, China). Fresh white pitayas (Hylocereus undulatus) were purchased from agricultural and sideline products wholesale center which is located in 183 Yisheng Road, Yuhangtang District, Hangzhou. The peels were removed and the juice was squeezed out of the fruits using a juice maker. The extracted juice was then centrifuged at 10,000 g for 40 min at 4°C. After that, the supernatant was filtered, collected and stored at -80°C. The content of bioactive compounds (e.g. total polyphenol and total flavonoids) in pitaya juice were determined using the methods described previously [19, 20].
Animals and diets All the protocols in this study were approved by the Committee on the Ethics of Animal Experiments of Zhejiang University (Permission Number: ZJU201550501) and the experimental procedures were performed according to the National Institutes of Health regulations for the care and use of animals in research. Moreover, all efforts were made to minimize suffering. Four-week-old C57BL/6J mice (male, n = 48) were supplied by the National Breeder Center of Rodents (Shanghai, China) and housed in a 12-hour light/dark cycle environment with free access to water and food and the temperature was kept at 23±3°C with a relative humidity of 50%±10%. After one week of acclimation, the mice were randomly assigned to the following four groups (n = 12): LFD, mice fed low-fat diet with free access to water, LFDJ, mice fed lowfat diet with free accesses to WPJ; HFD, mice fed high-fat diet with free access to water and HFDJ, mice fed high-fat diet with free access to WPJ. The ingredients and energy densities of the low-fat and high-fat diets are listed in the S1 Table. Juice and water were replaced every other day and the volumes consumed were recorded. The monitoring for body weight and
PLOS ONE | DOI:10.1371/journal.pone.0149670 February 25, 2016
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White Pitaya Juice Attenuates Insulin Resistance and Hepatic Steatosis
food consumption started in the first week and continued through the entire experiment. Food consumption of each dietary group was determined by weighing the total amount of food given on Monday and then subtracting by the amount of food remaining on Wednesday. The average food intake of each mouse was obtained by dividing the total number of the mice in the group. The low-fat diet (LFD) contained 3.85 kcal in each gram of food, and high-fat diet (HFD) contained 4.73 kcal in each gram of food. Based on the energy density and the amount of daily consumed food, the daily calorie intake was calculated. After 14 weeks of treatment, the mice were fasted overnight and sacrificed by decapitation. Blood samples were collected for serum preparation by centrifugation at 2000g for 15 minutes. The livers, hearts, kidneys, spleens, epididymal and perirenal fat and interscapular brown fat were collected, weighted and stored at -80°C prior to use.
Biochemical analysis Serum concentrations of glucose, TG, TC, ALT, AST, HDL-C and LDL-C were measured using an automatic biochemistry analyzer (ACCUTE TBA-40FR, Japan) according to the manufacturer’s instructions. The commercial ELISA assay kits were used to determine the serum levels of leptin (Cat#: MOB00), insulin (Cat#: E-EL-M2614c), adiponectin (Cat#: MRP300), LPS (Cat#: E-EL-0025c), FGF21 (Cat#: MF2100) and NPY (Cat#: E-EL-M0820c).
HOMA-IR and HOMA-IS analysis The homeostasis model assessment (HOMA) was applied to calculate the insulin resistance (HOMA-IR) and insulin sensitive index (HOMA-IS) in accordance with the following formulas: HOMA IR ¼ serum glucose ðmmol=LÞ serum insulin ðmUÞ = 22:5 HOMA IS ¼ 1 = ½serum glucose ðmmol=LÞ serum insulin ðmUÞ
Hepatic lipid profile analysis Liver total lipids were extracted using the Folch method with minor modifications [21]. Briefly, the liver tissues were homogenized with chloroform/methanol mixture to a final volume 15 times the volume of the sample (0.2 g in 3 ml of solvent mixture). After dispersion, the mixture was agitated for 20 min. Then the homogenate was centrifuged to recover the liquid phase. After that, the solvent was washed with 0.2 volume (0.2 ml for 1 ml) of 0.9% NaCl solution. After vortexing for some seconds, the mixture was centrifuged at 2000 rpm/min to separate the two phases. After centrifugation and removing of the upper phase, the lower chloroform phase containing total lipids was evaporated under a nitrogen stream and the lipids were resuspended using 1% Triton X-100 in chloroform. The extracting contents of TG and TC in livers were determined using commercially available kits (Elabscience, Cat#: E-EL-M2603c and E-EL-M2608c) according to the manufacturer's instructions.
Histological analysis Specimens of liver, epididymal and perirenal white adipose and interscapular brown adipose were cut and fixed in 10% buffered formalin. For oil red O staining, the liver tissues were embedded in Optimal Cutting Temperature gel and the air-dried 6μm thick sections were dipped in formalin and washed with 0.5% oil red O solution. For H&E staining, the liver and adipose tissues were embedded in paraffin, sliced at 6μm and stained with hematoxylin and eosin (H&E). The images were captured by Olympus CX41 camera (Japan). To evaluate
PLOS ONE | DOI:10.1371/journal.pone.0149670 February 25, 2016
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White Pitaya Juice Attenuates Insulin Resistance and Hepatic Steatosis
hepatic steatosis and the adipocyte size, the morphometric analysis was performed on 3 randomly selected sections at different depths for each mouse, and the representative sections were from six mice of each individual dietary group. The two softwares ImageJ and Photoshop were used for cell counting and the quantification of adipocyte size. Initial assessment of adipocyte size was performed under lower magnification (40×, containing> 300 cells) and confirmed under higher magnification (100×, containing> 300 cells; or 400×, containing>50 cells). The liver steatosis was scored as follows: 0, steatosis involving 66% of fatty hepatocytes [22].
RNA extraction and quantitative Real-Time PCR Analysis Total RNA was extracted from the liver with Trizol (Invitrogen Technologies, USA) according to the manufacturer’s instructions. Then the total RNA concentrations were measured by NanoDrop 2000 (Thermo Scientific, Wilmington, USA). 1μg total RNA was converted to cDNA using a reverse transcription kit (Takara, Cat# RR047A). 3 pooled RNA samples were from the n = 12 total samples in which 4 mice were pooled to generate one individual sample in each dietary group. The 20μl PCR reaction mixtures consisted of 1μl cDNA, paired-primers (300nM) and 10μl SYBR1 Green QPCR Master Mix (Roche). The amplification program performed on Applied Biosystems 7500 system was as follows: 3 min at 50°C, 10 min at 95°C, then 31 cycles of 15 s at 95°C and then 60s at 60°C followed by melting curve for 60s at 95°C, then gradual decrease to 50°C, 20 s at 50°C, then gradually increase to 95°C, and last 20 s. The primers are presented in the S2 Table. The relative expression of mRNA was normalized using the geometric mean of 5 housekeeping genes, including β-actin, RPS18, B2M, TBP and ARBP and the relative fold change was calculated using the 2-44Ct method.
Statistical analysis Values were expressed as means ± SEM. The statistical analyses were performed using SPSS 19.0 statistical software and the differences among multiple groups were assessed by one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test. p< 0.05 was considered statistically significant.
Results Characterization of white pitaya juice The bioactive compounds contained in WPJ were listed in Table 1. Table 1. Chemical characterization of white pitaya juice. Component
Amount (g / L)
Protein
8.26±0.25
Total sugar
60.42±4.16
Fiber Total polyphenols Total flavonoids Vitamin C
2.03±0.05 0.32±0.02 GAE 0.27±0.03 RE 0.08±0.004
GAE, gallic acid; RE, rutin. doi:10.1371/journal.pone.0149670.t001
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White Pitaya Juice Attenuates Insulin Resistance and Hepatic Steatosis
Effect of WPJ on body weight gain, tissue weights, food intake, liquid intake and serum lipid profile As depicted in Table 2, high-fat diet feeding induced a great body weight gain in mice, but no significant differences in either final body weight or the cumulative weight gain were observed between the HFDJ and HFD group. The similar results were found between LFD and LFDJ group. In addition, mice in LFD and LFDJ group consumed more food than those in HFD and HFDJ group, and mice in LFDJ and HFDJ group intake more liquid than those in LFD and HFD group. But there were no differences in the calorie intake among all the dietary groups. In addition, according to the FDA ratio of conversion, the human-equivalent dose for pitaya juice based on body surface area is approximately 440ml for a 60Kg human. High-fat diet-induced a Table 2. Body weights, cumulative weight gains, tissue weights and serum parameters in male C57BL/6J mice. Items
LFD
LFDJ
HFD
HFDJ
Initial BW (g)
19.18±0.47
19.48±0.38
19.74±0.64
18.76±0.51
Final BW (g)
27.31±0.73a
26.92±0.57a
46.32±0.85b
44.69±0.87b
b
25.93±0.66b
Cumulative weight gain (g)
a
8.14±0.91
a
26.58±0.99
a
b
7.44±0.68
a
Food intake (g/mouse/day)
3.09±0.08
3.19±0.08
2.54±0.09
2.53±0.07b
Calorie intake (kcal/mouse/day)
11.9±0.31
12.28±0.29
12±0.44
11.98±0.33
Liquid intake (ml/mouse/day)
2.99±0.08a
3.3±0.07b
2.61±0.06c
2.98±0.07a
Heart
0.15±0.01
0.16±0.01
0.16±0.01
0.17±0.02
Liver
1.04±0.06a
0.97±0.05a
1.44±0.06b
Tissue weight (g)
a
a
1.16±0.05a b
0.07±0.003c
Spleen
0.06±0.004
0.05±0.003
0.08±0.004
Kidney
a
a
0.33±0.01
b
0.44±0.02
0.38±0.02a
a
a
b
1.67±0.09c
Perirenal WAT
0.33±0.01 0.18±0.03
0.2±0.02
a
1.9±0.11 a
b
Epididymal WAT
0.41±0.07
0.39±0.08
2.67±0.15
2.08±0.1c
Interscapular BAT
0.15±0.01
0.15±0.02
0.22±0.04
0.18±0.02
0.57±0.03a
0.6±0.04a
0.34±0.01b
0.38±0.02b
Tissue index (% BW) Heart Liver Spleen Kidney Perirenal WAT
a
ab
3.8±0.21
3.64±0.24 a
0.21±0.01
0.2±0.01
a
1.23±0.06 0.65±0.1
0.16±0.01c
b
0.18±0.01
a
0.96±0.05
0.84±0.04b
a
b
3.75±0.23b
0.72±0.08 a
2.6±0.14c
bc
3.13±0.16
ab
1.22±0.06
a
b
4.1±0.21
a
b
Epididymal WAT
1.46±0.23
1.41±0.25
5.75±0.29
4.46±0.23c
Interscapular BAT
0.56±0.04
0.56±0.08
0.48±0.09
0.41±0.05
TG (mmol/L)
0.89±0.08a
0.68±0.06b
1.44±0.08c
1±0.07a
TC (mmol/L)
a
a
3.35±0.23
b
5.77±0.31
3.79±0.23a
a
a
b
4.58±0.13b
Serum parameters
HDL-C (mmol/L) LDL-C (mmol/L) AST (U/L) ALT (U/L)
3.09±0.29 2.61±0.18
2.78±0.2
a
5.01±0.3 a
0.41±0.02
b
0.37±0.03 a
139.8±8.94 a
39±2.93
137.7±10.19 a
27.3±2.18
0.28±0.03c
0.76±0.04 a
b
156.7±8.17a
b
57.8±7.71c
185.4±8.19 109.5±5.08
LFD, mice fed low-fat diet; LFDJ, mice fed low-fat diet with supplementation of WPJ; HFD, mice fed high-fat diet; HFDJ, mice fed high-fat diet with supplementation of WPJ. BW, body weight; WAT, white adipose; BAT, brown adipose; TG, triglyceride; TC, total cholesterol; ALT, Alanine aminotransferase; AST, aspartate aminotransferase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. Values are presented as means ± SEM (n = 10). Data not sharing a common superscript letter differ significantly among groups (p
White Pitaya (Hylocereus undatus) Juice Attenuates Insulin Resistance and Hepatic Steatosis in Diet-Induced Obese Mice Haizhao Song1,2, Zihuan Zheng1, Jianan Wu1, Jia Lai1, Qiang Chu1, Xiaodong Zheng1,2* 1 College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, P. R. China, 2 Fuli Institute of Food Science, Zhejiang University, Hangzhou, P. R. China * [email protected]
Abstract
OPEN ACCESS Citation: Song H, Zheng Z, Wu J, Lai J, Chu Q, Zheng X (2016) White Pitaya (Hylocereus undatus) Juice Attenuates Insulin Resistance and Hepatic Steatosis in Diet-Induced Obese Mice. PLoS ONE 11 (2): e0149670. doi:10.1371/journal.pone.0149670 Editor: Jonathan Peterson, East Tennessee State University, UNITED STATES Received: September 18, 2015 Accepted: February 1, 2016 Published: February 25, 2016 Copyright: © 2016 Song et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Key Technology R&D Program of China (Grant No.2012BAD33B08, http://program.most.gov.cn/, received by XDZ), Zhejiang Provincial Natural Science Foundation of China (Grant No. Z14C200006, http://www.zjnsf.gov.cn/, received by XDZ) and the Foundation of Fuli Institute of Food Science, Zhejiang University (Grant No. KY201301, http://fifs.zju.edu.cn/content/detail.php?sid=3&cid= 642, received by XDZ). The funders had no role in
Insulin resistance and hepatic steatosis are the most common complications of obesity. Pitaya is an important source of phytochemicals such as polyphenols, flavonoid and vitamin C which are related to its antioxidant activity. The present study was conducted to evaluate the influence of white pitaya juice (WPJ) on obesity-related metabolic disorders (e.g. insulin resistance and hepatic steatosis) in high-fat diet-fed mice. Forty-eight male C57BL/6J mice were assigned into four groups and fed low-fat diet with free access to water or WPJ, or fed high-fat diet with free access to water or WPJ for 14 weeks. Our results showed that administration of WPJ improved high-fat diet-induced insulin resistance, hepatic steatosis and adipose hypertrophy, but it exerted no influence on body weight gain in mice. Hepatic gene expression analysis indicated that WPJ supplement not only changed the expression profile of genes involved in lipid and cholesterol metabolism (Srebp1, HMGCoR, Cpt1b, HL, Insig1 and Insig2) but also significantly increased the expression levels of FGF21-related genes (Klb, FGFR2, Egr1 and cFos). In conclusion, WPJ protected from diet-induced hepatic steatosis and insulin resistance, which was associated with the improved FGF21 resistance and lipid metabolism.
Introduction Type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) are common consequences of a combination of metabolic disorders such as dyslipidemia, hepatic steatosis, glucose intolerance and insulin resistance [1]. Mounting evidence suggests that insulin resistance and hepatic steatosis, the most common complications of obesity, are characterized by impaired energy metabolism and attributed by complex interactions between genetic background and environmental impacts such as dietary and sedentary habits[2–6]. Moreover, insulin resistance is often accompanied by hepatic steatosis [7]. Traditional pharmacotherapies for type 2 diabetes and NAFLD are often accompanied by side effects. Recently, growing evidence has suggested that natural bioactive compounds in food exerts many beneficial effects on obesity and its metabolic consequences [8–10].
PLOS ONE | DOI:10.1371/journal.pone.0149670 February 25, 2016
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White Pitaya Juice Attenuates Insulin Resistance and Hepatic Steatosis
study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Pitaya, also known as dragon fruit, is among the most important commercial tropical fruits around the world, and three varieties have been classified as Hylocereu polyrhizus (red pitaya), Hylocereus undatus (white pitaya) and Hylocereus megalanthus (Yellow pitaya) [11]. Pitaya is an important source of phytochemicals such as polyphenols, flavonoid and vitamin C which are related to its antioxidant activity [12, 13]. Especially the red and white pitaya have recently drawn growing attention worldwide not only because of their economic values but also their health benefits [14]. Red pitaya consumption was reported to decrease total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels while increasing the high-density lipoprotein cholesterol (HDL-C) levels in type 2 diabetic subjects. The similar lipid-improving effect of its menthol extract was found in high-fat diet-fed rats [15, 16]. In addition, the antidiabetic effect of red pitaya has also recently been demonstrated. For instance, red pitaya significantly improved insulin resistance in rats and 600 g amount of red pitaya fruit consumption every day decreased the blood glucose level in type II diabetic subjects [15, 17]. Ramli et al. also reported that supplementation of red pitaya juice could induce a trend of glucose and uric acid normalization associated with reduced ALP and ALT but increased AST levels in high-carbohydrate and high-fat diet-fed rats [18].Though both red and white pitaya are reported to be rich, natural and cost-effective source of bioactive nutrients, few studies focused on the beneficial effects of white pitaya on diabetes and NAFLD. The objective of the present study was to investigate the influence of white pitaya juice (WPJ) on obesity-related hepatic steatosis and insulin resistance in high-fat diet-induced obese mice and explore the underlying mechanism by which pitaya juice exerted its beneficial effects. To the best of our knowledge, this is the first study to evaluate the effect of white pitaya on type II diabetes and NAFLD.
Materials and Methods Chemical reagents and juice preparation The solvents and reagents were obtained from Aladdin (Shanghai, China). Fresh white pitayas (Hylocereus undulatus) were purchased from agricultural and sideline products wholesale center which is located in 183 Yisheng Road, Yuhangtang District, Hangzhou. The peels were removed and the juice was squeezed out of the fruits using a juice maker. The extracted juice was then centrifuged at 10,000 g for 40 min at 4°C. After that, the supernatant was filtered, collected and stored at -80°C. The content of bioactive compounds (e.g. total polyphenol and total flavonoids) in pitaya juice were determined using the methods described previously [19, 20].
Animals and diets All the protocols in this study were approved by the Committee on the Ethics of Animal Experiments of Zhejiang University (Permission Number: ZJU201550501) and the experimental procedures were performed according to the National Institutes of Health regulations for the care and use of animals in research. Moreover, all efforts were made to minimize suffering. Four-week-old C57BL/6J mice (male, n = 48) were supplied by the National Breeder Center of Rodents (Shanghai, China) and housed in a 12-hour light/dark cycle environment with free access to water and food and the temperature was kept at 23±3°C with a relative humidity of 50%±10%. After one week of acclimation, the mice were randomly assigned to the following four groups (n = 12): LFD, mice fed low-fat diet with free access to water, LFDJ, mice fed lowfat diet with free accesses to WPJ; HFD, mice fed high-fat diet with free access to water and HFDJ, mice fed high-fat diet with free access to WPJ. The ingredients and energy densities of the low-fat and high-fat diets are listed in the S1 Table. Juice and water were replaced every other day and the volumes consumed were recorded. The monitoring for body weight and
PLOS ONE | DOI:10.1371/journal.pone.0149670 February 25, 2016
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White Pitaya Juice Attenuates Insulin Resistance and Hepatic Steatosis
food consumption started in the first week and continued through the entire experiment. Food consumption of each dietary group was determined by weighing the total amount of food given on Monday and then subtracting by the amount of food remaining on Wednesday. The average food intake of each mouse was obtained by dividing the total number of the mice in the group. The low-fat diet (LFD) contained 3.85 kcal in each gram of food, and high-fat diet (HFD) contained 4.73 kcal in each gram of food. Based on the energy density and the amount of daily consumed food, the daily calorie intake was calculated. After 14 weeks of treatment, the mice were fasted overnight and sacrificed by decapitation. Blood samples were collected for serum preparation by centrifugation at 2000g for 15 minutes. The livers, hearts, kidneys, spleens, epididymal and perirenal fat and interscapular brown fat were collected, weighted and stored at -80°C prior to use.
Biochemical analysis Serum concentrations of glucose, TG, TC, ALT, AST, HDL-C and LDL-C were measured using an automatic biochemistry analyzer (ACCUTE TBA-40FR, Japan) according to the manufacturer’s instructions. The commercial ELISA assay kits were used to determine the serum levels of leptin (Cat#: MOB00), insulin (Cat#: E-EL-M2614c), adiponectin (Cat#: MRP300), LPS (Cat#: E-EL-0025c), FGF21 (Cat#: MF2100) and NPY (Cat#: E-EL-M0820c).
HOMA-IR and HOMA-IS analysis The homeostasis model assessment (HOMA) was applied to calculate the insulin resistance (HOMA-IR) and insulin sensitive index (HOMA-IS) in accordance with the following formulas: HOMA IR ¼ serum glucose ðmmol=LÞ serum insulin ðmUÞ = 22:5 HOMA IS ¼ 1 = ½serum glucose ðmmol=LÞ serum insulin ðmUÞ
Hepatic lipid profile analysis Liver total lipids were extracted using the Folch method with minor modifications [21]. Briefly, the liver tissues were homogenized with chloroform/methanol mixture to a final volume 15 times the volume of the sample (0.2 g in 3 ml of solvent mixture). After dispersion, the mixture was agitated for 20 min. Then the homogenate was centrifuged to recover the liquid phase. After that, the solvent was washed with 0.2 volume (0.2 ml for 1 ml) of 0.9% NaCl solution. After vortexing for some seconds, the mixture was centrifuged at 2000 rpm/min to separate the two phases. After centrifugation and removing of the upper phase, the lower chloroform phase containing total lipids was evaporated under a nitrogen stream and the lipids were resuspended using 1% Triton X-100 in chloroform. The extracting contents of TG and TC in livers were determined using commercially available kits (Elabscience, Cat#: E-EL-M2603c and E-EL-M2608c) according to the manufacturer's instructions.
Histological analysis Specimens of liver, epididymal and perirenal white adipose and interscapular brown adipose were cut and fixed in 10% buffered formalin. For oil red O staining, the liver tissues were embedded in Optimal Cutting Temperature gel and the air-dried 6μm thick sections were dipped in formalin and washed with 0.5% oil red O solution. For H&E staining, the liver and adipose tissues were embedded in paraffin, sliced at 6μm and stained with hematoxylin and eosin (H&E). The images were captured by Olympus CX41 camera (Japan). To evaluate
PLOS ONE | DOI:10.1371/journal.pone.0149670 February 25, 2016
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White Pitaya Juice Attenuates Insulin Resistance and Hepatic Steatosis
hepatic steatosis and the adipocyte size, the morphometric analysis was performed on 3 randomly selected sections at different depths for each mouse, and the representative sections were from six mice of each individual dietary group. The two softwares ImageJ and Photoshop were used for cell counting and the quantification of adipocyte size. Initial assessment of adipocyte size was performed under lower magnification (40×, containing> 300 cells) and confirmed under higher magnification (100×, containing> 300 cells; or 400×, containing>50 cells). The liver steatosis was scored as follows: 0, steatosis involving 66% of fatty hepatocytes [22].
RNA extraction and quantitative Real-Time PCR Analysis Total RNA was extracted from the liver with Trizol (Invitrogen Technologies, USA) according to the manufacturer’s instructions. Then the total RNA concentrations were measured by NanoDrop 2000 (Thermo Scientific, Wilmington, USA). 1μg total RNA was converted to cDNA using a reverse transcription kit (Takara, Cat# RR047A). 3 pooled RNA samples were from the n = 12 total samples in which 4 mice were pooled to generate one individual sample in each dietary group. The 20μl PCR reaction mixtures consisted of 1μl cDNA, paired-primers (300nM) and 10μl SYBR1 Green QPCR Master Mix (Roche). The amplification program performed on Applied Biosystems 7500 system was as follows: 3 min at 50°C, 10 min at 95°C, then 31 cycles of 15 s at 95°C and then 60s at 60°C followed by melting curve for 60s at 95°C, then gradual decrease to 50°C, 20 s at 50°C, then gradually increase to 95°C, and last 20 s. The primers are presented in the S2 Table. The relative expression of mRNA was normalized using the geometric mean of 5 housekeeping genes, including β-actin, RPS18, B2M, TBP and ARBP and the relative fold change was calculated using the 2-44Ct method.
Statistical analysis Values were expressed as means ± SEM. The statistical analyses were performed using SPSS 19.0 statistical software and the differences among multiple groups were assessed by one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test. p< 0.05 was considered statistically significant.
Results Characterization of white pitaya juice The bioactive compounds contained in WPJ were listed in Table 1. Table 1. Chemical characterization of white pitaya juice. Component
Amount (g / L)
Protein
8.26±0.25
Total sugar
60.42±4.16
Fiber Total polyphenols Total flavonoids Vitamin C
2.03±0.05 0.32±0.02 GAE 0.27±0.03 RE 0.08±0.004
GAE, gallic acid; RE, rutin. doi:10.1371/journal.pone.0149670.t001
PLOS ONE | DOI:10.1371/journal.pone.0149670 February 25, 2016
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White Pitaya Juice Attenuates Insulin Resistance and Hepatic Steatosis
Effect of WPJ on body weight gain, tissue weights, food intake, liquid intake and serum lipid profile As depicted in Table 2, high-fat diet feeding induced a great body weight gain in mice, but no significant differences in either final body weight or the cumulative weight gain were observed between the HFDJ and HFD group. The similar results were found between LFD and LFDJ group. In addition, mice in LFD and LFDJ group consumed more food than those in HFD and HFDJ group, and mice in LFDJ and HFDJ group intake more liquid than those in LFD and HFD group. But there were no differences in the calorie intake among all the dietary groups. In addition, according to the FDA ratio of conversion, the human-equivalent dose for pitaya juice based on body surface area is approximately 440ml for a 60Kg human. High-fat diet-induced a Table 2. Body weights, cumulative weight gains, tissue weights and serum parameters in male C57BL/6J mice. Items
LFD
LFDJ
HFD
HFDJ
Initial BW (g)
19.18±0.47
19.48±0.38
19.74±0.64
18.76±0.51
Final BW (g)
27.31±0.73a
26.92±0.57a
46.32±0.85b
44.69±0.87b
b
25.93±0.66b
Cumulative weight gain (g)
a
8.14±0.91
a
26.58±0.99
a
b
7.44±0.68
a
Food intake (g/mouse/day)
3.09±0.08
3.19±0.08
2.54±0.09
2.53±0.07b
Calorie intake (kcal/mouse/day)
11.9±0.31
12.28±0.29
12±0.44
11.98±0.33
Liquid intake (ml/mouse/day)
2.99±0.08a
3.3±0.07b
2.61±0.06c
2.98±0.07a
Heart
0.15±0.01
0.16±0.01
0.16±0.01
0.17±0.02
Liver
1.04±0.06a
0.97±0.05a
1.44±0.06b
Tissue weight (g)
a
a
1.16±0.05a b
0.07±0.003c
Spleen
0.06±0.004
0.05±0.003
0.08±0.004
Kidney
a
a
0.33±0.01
b
0.44±0.02
0.38±0.02a
a
a
b
1.67±0.09c
Perirenal WAT
0.33±0.01 0.18±0.03
0.2±0.02
a
1.9±0.11 a
b
Epididymal WAT
0.41±0.07
0.39±0.08
2.67±0.15
2.08±0.1c
Interscapular BAT
0.15±0.01
0.15±0.02
0.22±0.04
0.18±0.02
0.57±0.03a
0.6±0.04a
0.34±0.01b
0.38±0.02b
Tissue index (% BW) Heart Liver Spleen Kidney Perirenal WAT
a
ab
3.8±0.21
3.64±0.24 a
0.21±0.01
0.2±0.01
a
1.23±0.06 0.65±0.1
0.16±0.01c
b
0.18±0.01
a
0.96±0.05
0.84±0.04b
a
b
3.75±0.23b
0.72±0.08 a
2.6±0.14c
bc
3.13±0.16
ab
1.22±0.06
a
b
4.1±0.21
a
b
Epididymal WAT
1.46±0.23
1.41±0.25
5.75±0.29
4.46±0.23c
Interscapular BAT
0.56±0.04
0.56±0.08
0.48±0.09
0.41±0.05
TG (mmol/L)
0.89±0.08a
0.68±0.06b
1.44±0.08c
1±0.07a
TC (mmol/L)
a
a
3.35±0.23
b
5.77±0.31
3.79±0.23a
a
a
b
4.58±0.13b
Serum parameters
HDL-C (mmol/L) LDL-C (mmol/L) AST (U/L) ALT (U/L)
3.09±0.29 2.61±0.18
2.78±0.2
a
5.01±0.3 a
0.41±0.02
b
0.37±0.03 a
139.8±8.94 a
39±2.93
137.7±10.19 a
27.3±2.18
0.28±0.03c
0.76±0.04 a
b
156.7±8.17a
b
57.8±7.71c
185.4±8.19 109.5±5.08
LFD, mice fed low-fat diet; LFDJ, mice fed low-fat diet with supplementation of WPJ; HFD, mice fed high-fat diet; HFDJ, mice fed high-fat diet with supplementation of WPJ. BW, body weight; WAT, white adipose; BAT, brown adipose; TG, triglyceride; TC, total cholesterol; ALT, Alanine aminotransferase; AST, aspartate aminotransferase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. Values are presented as means ± SEM (n = 10). Data not sharing a common superscript letter differ significantly among groups (p
